Recent advances in molecular biology have greatly accelerated the rate at which genes can be cloned and characterized, rendering determination of the complete genomic sequence of an organism an attainable goal. Accordingly, large scale genomic sequencing efforts for several organisms including bacteria, yeast, and nematodes have already yielded extensive genomic sequence information. Recently, the Human Genome Project, an ambitious project to obtain the entire sequence of the human genome, has commenced. The Human Genome Project paves the way for an even more ambitious project sometimes referred to as the Human Genome Diversity Project, a project aimed at identifying and characterizing allelic differences between humans which manifest themselves in a phenotypically important manner. For example, some allelic variations may be the source of debilitating diseases such as sickle cell anemia or neoplastic diseases.
With cloning techniques well developed, the rate at which information can be extracted from the cloned DNA becomes a limiting factor in determining the genomic sequence of an organism. Accordingly, advances in automated sequencing will reduce the cost and time required to sequence the genomes of model organisms.
For example, in order to sequence the 3 billion nucleotides in the human genome in a ten year span, it is necessary for the automated sequencing devices to achieve a sequencing rate of three hundred million bases per year. At the same time, the sequence information obtained using these automated systems must be accurate, reliable, and efficient without requiring the involvement of highly skilled personnel to a high degree. In addition, the cost of operating and maintaining the automated sequencing devices must be minimized.
In the past, slab gel electrophoresis was used to sequence DNA. (See U.S. Pat. No. 4,811,218 and EPO 0533302A1, the disclosures of which are incorporated herein by reference). However, such techniques are prohibitively limited in the context of genomic sequencing efforts. Recently, capillary electrophoresis has emerged as a viable approach to genomic sequencing. In capillary electrophoresis the design products of sequencing reactions conducted on the nucleic acids to be sequenced are applied to small diameter capillary tubes containing a separating medium such as a soluble cellulose derivative or polyacrylamide. A high voltage is applied along the tubes, thereby causing the nucleic acids to migrate along the length of the capillary tubes. As in conventional sequencing techniques, the differential migration rates of nucleic acids of different lengths enables sequence determination. Nucleic acids migrating through the capillary tubes are detected upon reaching a detection region in the capillary tubes using such techniques as laser induced fluorescence.
While capillary electrophoresis permits high resolution of nucleic acids of different lengths and rapid sequence determination, several technical hurdles remain in the application of this technology to genomic sequencing efforts. One important limitation in existing methods is the difficulty in obtaining sequence information from a large number of capillaries simultaneously.
Huang et al. provided a device in which multiple capillaries are arrayed side by side as illustrated in FIG. 1, and are sequentially scanned by a laser and fluorescence is detected using a photomultiplier. (See Huang et al., Anal. Chem. 64:967-972 (1992), the disclosure of which is incorporated herein by reference). However, the effectiveness of the device of Huang is reduced as a result of lightscatter from the capillary walls and the interfaces between the separation medium and the capillaries. Furthermore, in the Huang device, the entire stage on which the capillaries are mounted is linearly translated back and forth underneath the light illumination and collection apparatus, resulting in stress on the capillaries and difficulties in precise position control.
Dovichi et al. provided a device in which multiple rows of capillaries terminate at different levels in a sheath flow cuvette. (See WO 94/29712, the disclosure of which is incorporated herein by reference). Sheath fluid draws individual sample streams through the cuvette. However, the device of Dovichi et al. requires a bubble removing system to ensure that bubbles do not form in the cuvette. To reduce background signal the Dovichi device requires the use of highly purified sheath fluid. In addition, in order to achieve the required sensitivity of signal detection, the Dovichi design requires placement of the laser very close to the termini of the capillaries. Finally, with the Dovichi system it is difficult to adjust the system after each use and to change the capillaries.
For the preceding reasons, there is a need for a detection system which achieves a high throughput while requiring little attention by highly trained personnel.